Mitochondrial (mt) are central organelles with activities in energetics as well as in a number of pathways linked to celular life, disease, ageing and death. Of endosymbiotic origin, mammalian mt have kept only a limlted genome (16.5 Kb) coding for 13 proteins (all sub-units of the respiratory chain complexes), 2 rRNAs and 22 tRNAs. All expressed RNAs are partially degenerated as compared to their bacterial ancestors. Other actors of the translation machinery, including aminoacyl-tRNA synthetases (aaRSs), are nuclear-encoded and imported from the cytosol to the mitochondria thanks to the presence of a mitochondrial targeting sequence (MTS). The mt genome still undergoes a high mutation rate with tRNA genes as hot spots for pathogenic mutations (more than 130 identified so far, see here) correlated to a diversity of neurodegenerative and neuromuscular disorders (list of commonly used abbreviations and name of related pathologies). Our long-term major goal is to decipher structure/function relationships of macromolecules forming the human (and by extension the mammalian) mt translation machinery as well in healthy as in pathological contexts, with major focus on aminoacylation systems. The aims are to understand the specific functional properties and evolutionary histories of the latter, to explore new functions, and to investigatie molecular dysfunction in human disorders. Approaches combin biochemistry, molecular and celluar biology, bioinformatics, biophysics and structural biology.

tRNA databases, tertiary structural networks and deep phylogeny

Our previous study have highlighted strong structural degeneration of mammalian mt-tRNAs as compared to canonical tRNAs, and raised the question of tertiary interaction networks allowing functional molecules. Chemical and enzymatic structure probing in solution combined to 3D modeling, revealed that a typical human mt-tRNA (namely tRNAAsp) has lost D/T-loops interactions but still maintains the full set of tertiary interactions forming the core of the 3D structure. These rules hold true for most mt-tRNAAsp from our Mamit-tRNA database when screened for conservation of Leontis-Westhof interactions rather than for simple nucleotide conservation. Implementation of bioinformatic automated search tool for the 3D interaction network in any mt-tRNAs is now in progress. A new mt-tRNA annotation tool enabling rigorous detection of non-classical tRNAs has been developped (MITOS). Its application to mt-genome annotation and organization allows for deep phylogeny investigation (collaboration: P. Stadler, Leipzig). We are also in charge of the world tRNA compilation database (tRNAdb2009) with updates and complete reimplementation as a relational searchable and user-friendly database.

Human mt-tRNAAsp 3D folding is compatible with the existence of "classical" tertiary interaction network forming the core of the molecule, but misses D- and T-loop interactions. In the right panel, the CCA-end points towards the reader. Bottom: detailed views on the individual core interactions. The six tertiary interactions found in classical tRNAs are highlighted in the model, in agreement with the chemical probing data and conservation of Leontis-Westhof rules in 136 mammalian mt-tRNAAsp.

Biocrystallogenesis and mt-aaRSs crystal structures

Crystallography is major investigation tool to study the architecture of target macromoleucles. Since obtaining high-quality crystals is still a major bottleneck, we have strongly contributed to the world-wide effort to promote the use of (i) phase diagrams, (ii) gelled media, and (iii) microfluidics to define and optimize appropriate crystallization conditions (see figure). These approaches have been directly applied to the benefit of proper or collaborative structural biology projects including the deciphering of crystallographic structures of human mt-TyrRS and mt-AspRS. Dynamic light scattering (DLS) has become a systematic quality control tool of investigated macromolecules and allows to monitor formation of macromolecular complexes in solution. Insight into dynamics could be gained by comparing crystal polymorphs capturing different conformational states.

Despite its bacterial origin, with 43% identity with the E. coli enzyme, human mt-AspRS displays distinctive properties. First, it specifically aminoacylates its cognate tRNA in spite of the absence of major aspartylation identity elements, as a result of combined co-evolution of the synthetase and rapid degeneration of the mt-tRNA. A systematic bioinformatic search for aminoacylation identity sets in mammalian mt-tRNAs, based on modern discrete mathematical methods (BOOL-AN), is in progress (coll. E. Jako, Budapest). Second, this enzyme cross-aminoacylates aspartate tRNAs from various sources while E. coli AspRS is only active on its natural substrate, in-line with the general trend of unilateral aminoacylation by bacterial aaRS as opposed to large substrate tolerance of mt-aaRSs. A progressive mutational attempt to convert the mt-tRNA into an efficient substrate for the bacterial enzyme revealed not only requirements of a full identity set, but also of numerous structural constraints. Third, the two synthetases express a different sensitivity to non-hydrolysable adenylate analogs. Human mt-AspRS is more sensitive to both L-aspartol-adenylate and 5’-O-[N-(L-aspartyl)sulfamoyl]adenosine inhibitors as compared to bacterial enzymes. This is not correlated with clear-cut structural features in the catalytic site as deduced from docking experiments, but may result from dynamic properties. Insight into these functional differences between the mt- and bacterial synthetases were further tackled by using biophysical approaches. Isothermal calorimetry (ITC) revealed that tRNA/aaRS complexes are driven by differential thermodynamic parameters. Further, the bacterial and mt enzymes differ by their thermostability monitored by DLS.

Human mt aminoacylation systems and neurodegeneration

For over 20 years, mt-disorders were solely correlated to tRNA gene mutations. Mutations lead to a mosaic of molecular effects, with recurrent impact on tRNA structure. In 2007, we contributed to the first description of leukoencephalopathies caused by mutations in the nuclear gene of a mt-aaRS (some mutations found in the gene coding for the mt-AspRS are displayed on a 3D representation of the enzyme, left hand pannel of the figure). The single mutation located in the predicted mt-targeting signal impairs the import process of the protein across mt-membranes. The other mutations do not impact aminoacylation efficiency, suggesting that alternative functions of the protein are affected. As a consequence, partners of mt-aaRS were searched using immunoprecipitation and tap-tagging and the reality of splice-variants of mt-aaRSs has been explored. A candidate mt-AspRS mRNA, lacking one exon in frame, is present in significant quantities in 20 tested human tissues and the corresponding protein is immunodetected in skeletal muscle biopsy. Finally, the intriguing tissue specificity of mt-disorders has been approached. The evaluation by qPCR of 19 mt-aaRSs mRNA and of mt-16S rRNA levels in 20 different tissues (right hand pannel of the figure) led to a highly variable landscape, with lowest mRNA levels for highest mt-translation activities. This suggests that the high sensitivity of brain, heart and muscle to mt-tRNA or mt-aaRS mutations may actually result from even weak dysfunction of intrinsically limiting amounts of aaRS, key actors of translation.